PKD is a kinase of Vps34 that mediates ROS-induced autophagy downstream of DAPk

Autophagy, a process in which cellular components are engulfed and degraded within double-membrane vesicles termed autophagosomes, has an important role in the response to oxidative damage. Here we identify a novel cascade of phosphorylation events, involving a network of protein and lipid kinases, as crucial components of the signaling pathways that regulate the induction of autophagy under oxidative stress. Our findings show that both the tumor-suppressor death-associated protein kinase (DAPk) and protein kinase D (PKD), which we previously showed to be phosphorylated and consequently activated by DAPk, mediate the induction of autophagy in response to oxidative damage. Furthermore, we map the position of PKD within the autophagic network to Vps34, a lipid kinase whose function is indispensable for autophagy, and demonstrate that PKD is found in the same molecular complex with Vps34. PKD phosphorylates Vps34, leading to activation of Vps34, phosphatydilinositol-3-phosphate (PI(3)P) formation, and autophagosome formation. Consistent with its identification as a novel inducer of the autophagic machinery, we show that PKD is recruited to LC3-positive autophagosomes, where it localizes specifically to the autophagosomal membranes. Taken together, our results describe PKD as a novel Vps34 kinase that functions as an effecter of autophagy under oxidative stress

Practice patterns of naturopathic physicians: results from a random survey of licensed practitioners in two US States

BACKGROUND: Despite the growing use of complementary and alternative medicine (CAM) by consumers in the U.S., little is known about the practice of CAM providers. The objective of this study was to describe and compare the practice patterns of naturopathic physicians in Washington State and Connecticut. METHODS: Telephone interviews were conducted with state-wide random samples of licensed naturopathic physicians and data were collected on consecutive patient visits in 1998 and 1999. The main outcome measures were: Sociodemographic, training and practice characteristics of naturopathic physicians; and demographics, reasons for visit, types of treatments, payment source and visit duration for patients. RESULT: One hundred and seventy practitioners were interviewed and 99 recorded data on a total of 1817 patient visits. Naturopathic physicians in Washington and Connecticut had similar demographic and practice characteristics. Both the practitioners and their patients were primarily White and female. Almost 75% of all naturopathic visits were for chronic complaints, most frequently fatigue, headache, and back symptoms. Complete blood counts, serum chemistries, lipids panels and stool analyses were ordered for 4% to 10% of visits. All other diagnostic tests were ordered less frequently. The most commonly prescribed naturopathic therapeutics were: botanical medicines (51% of visits in Connecticut, 43% in Washington), vitamins (41% and 43%), minerals (35% and 39%), homeopathy (29% and 19%) and allergy treatments (11% and 13%). The mean visit length was about 40 minutes. Approximately half the visits were paid directly by the patient. CONCLUSION: This study provides information that will help other health care providers, patients and policy makers better understand the nature of naturopathic care

JNK modulates FOXO3a for the expression of the mitochondrial death and mitophagy marker BNIP3 in pathological hypertrophy and in heart failure

Bcl-2 E1B 19-KDa interacting protein 3 (BNIP3) is a mitochondrial death and mitophagy marker, which is involved in inducing cardiac remodeling post myocardial infarction. In this study, we show that BNIP3 expression increases in stressed cardiomyocytes in vitro and in response to pressure overload in vivo, and that its transcription is directly related to JNK activity. BNIP3 expression gradually increased in the first weeks after pressure overload and peaked at the heart failure stage. Ultrastructurally, the mitochondrial area was inversely proportional to BNIP3 expression. Both JNK and AKT activities increased with pressure overload; however, JNK signaling dominated over AKT signaling for the activation of the transcription factor FOXO3a and for the transcription of its effector, BNIP3. 3-methyladenine attenuated JNK signaling and significantly decreased BNIP3 expression and reversed cardiac remodeling in heart failure. Ultrastructurally, the mitochondrial area was significantly increased in the 3-methyladenine group compared with placebo. Moreover, adenoviral gene delivery of dominant negative JNK in a rat model of pressure overload hypertrophy abolished the increase in BNIP3 expression in response to pressure overload. These results suggest that JNK signaling is a critical modulator of the transcription factor FOXO3a driving the expression of its effector, BNIP3, in heart failure and that JNK, through BNIP3, induces mitochondrial apoptosis and mitophagy

The Recognition of N-Glycans by the Lectin ArtinM Mediates Cell Death of a Human Myeloid Leukemia Cell Line

ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment

Androgens modulate autophagy and cell death via regulation of the endoplasmic reticulum chaperone glucose-regulated protein 78/BiP in prostate cancer cells

Pro-survival signalling mediated by the androgen receptor (AR) is implicated as a key contributor to prostate carcinogenesis. As prostate tumours are characterized by nutrient-poor, hypoxic and acidified microenvironments, one mechanism whereby AR signalling may contribute to survival is by promoting adaptation to cellular stress. Here we have identified a novel role for AR in the inhibition of autophagy induced by serum withdrawal. This blockade is attributed to AR-mediated upregulation of the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78/BiP (Grp78/BiP), and occurs independently of ER stress response pathway activation. Interestingly, AR activation did not affect serum starvation-induced mammalian target of rapamycin inhibition, illustrating that the adaptive role for androgens lies not in the ability to modulate nutrient sensing, but in the promotion of ER stability. Finally, we show that the adaptive advantage conferred by AR-mediated Grp78/BiP upregulation is temporary, as upon chronic serum starvation, AR activation delayed but did not suppress the onset of autophagy and cell death. This study reveals a novel mechanism whereby maintained AR signalling promotes temporary adaptation to cellular stress and in turn may contribute to the evasion of prostate tumour cell death

The conservation and uniqueness of the caspase family in the basal chordate, amphioxus

<p>Abstract</p> <p>Background</p> <p>The caspase family, which plays a central role in apoptosis in metazoans, has undergone an expansion in amphioxus, increasing to 45 members through domain recombination and shuffling.</p> <p>Results</p> <p>In order to shed light on the conservation and uniqueness of this family in amphioxus, we cloned three representative caspase genes, designated as <it>bbtCaspase-8, bbtCaspase-1/2 </it>and <it>bbtCaspase3</it>-like, from the amphioxus <it>Branchiostoma belcheri tsingtauense</it>. We found that <it>bbtCaspase-8 </it>with conserved protein architecture is involved in the Fas-associated death domain-Caspase-8 mediated pro-apoptotic extrinsic pathway, while <it>bbtCaspase3</it>-like may mediate a nuclear apoptotic pathway in amphioxus. Also, <it>bbtCaspase-1/2 </it>can co-localize with <it>bbtFADD2 </it>in the nucleus, and be recruited to the cytoplasm by amphioxus apoptosis associated speck-like proteins containing a caspase recruitment domain, indicating that <it>bbtCaspase-1/2 </it>may serve as a switch between apoptosis and caspase-dependent innate immune response in invertebrates. Finally, amphioxus extrinsic apoptotic pathway related caspases played important roles in early embryogenesis.</p> <p>Conclusions</p> <p>Our study not only demonstrates the conservation of <it>bbtCaspase-8 </it>in apoptosis, but also reveals the unique features of several amphioxus caspases with novel domain architectures arose some 500 million years ago.</p

Differential Regulation of Cutaneous Oncoprotein HPVE6 by wtp53, Mutant p53R248W and ΔNp63α is HPV Type Dependent

UV exposure and p53 mutations are major factors in non-melanoma skin cancer, whereas a role for HPV infections has not been defined. Previous data demonstrated the wtp53-mediated degradation of cutaneous HPV20E6 by caspase-3. ΔNp63α and hot-spot mutant p53R248W conveyed a protective effect on HPV20E6 under these conditions. We demonstrate a differential regulation by wtp53 of the E6 genes of cutaneous types HPV4, HPV5, HPV7, HPV27, HPV38, HPV48, HPV60 and HPV77. Caspase- or proteasome-mediated down-regulation was HPV type dependent. Mutant p53R248W up-regulated expression of all these E6 proteins as did ΔNp63α except for HPV38E6 which was down-regulated by the latter. None of these cellular proteins affected HPV41E6 expression. Ectopic expression of both mutp53R248W and ΔNp63α in the normal NIKS keratinocyte cell line harbouring endogenous p53 and p63however led to a down-regulation of HPV20E6. We demonstrate that HPV20E6 expression in these cells is modulated by additional, yet unidentified, cellular protein(s), which are not necessarily involved in apoptosis or autophagy. We further demonstrate proliferation of HPV20E6-expressing keratinocytes. Levels of proteins involved in cell cycle control, cyclin-D1, cdk6 and p16INK4a, phosphorylated pRB, as well as c-Jun and p-c-Jun, were all increased in these cells. HPV20E6 did not compete for the interaction between p16INK4a with cyclin-D1 or cdk6. Phosphorylation of pRB in the HPV20E6 expressing cells seems to be sufficient to override the cytokenetic block induced by the p16INK4a/pRB pathway. The present study demonstrates the diverse influence of p53 family members on individual cutaneous HPVE6 proteins. HPV20E6 expression also resulted in varying protein levels of factors involved in proliferation and differentiation

Mycobacterium tuberculosis Eis Regulates Autophagy, Inflammation, and Cell Death through Redox-dependent Signaling

The “enhanced intracellular survival” (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner

Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment

Cancer cells show a broad spectrum of bioenergetic states, with some cells using aerobic glycolysis while others rely on oxidative phosphorylation as their main source of energy. In addition, there is mounting evidence that metabolic coupling occurs in aggressive tumors, between epithelial cancer cells and the stromal compartment, and between well-oxygenated and hypoxic compartments. We recently showed that oxidative stress in the tumor stroma, due to aerobic glycolysis and mitochondrial dysfunction, is important for cancer cell mutagenesis and tumor progression. More specifically , increased autophagy/mitophagy in the tumor stroma drives a form of parasitic epithelial-stromal metabolic coupling. These findings explain why it is effective to treat tumors with either inducers or inhibitors of autophagy, as both would disrupt this energetic coupling. We also discuss evidence that glutamine addiction in cancer cells produces ammonia via oxidative mitochondrial metabolism. Ammonia production in cancer cells, in turn, could then help maintain autophagy in the tumor stromal compartment. In this vicious cycle, the initial glutamine provided to cancer cells would be produced by autophagy in the tumor stroma. Thus, we believe that parasitic epithelial-stromal metabolic coupling has important implications for cancer diagnosis and therapy, for example, in designing novel metabolic imaging techniques and establishing new targeted therapies. In direct support of this notion, we identified a loss of stromal caveolin-1 as a marker of oxidative stress, hypoxia, and autophagy in the tumor microenvironment, explaining its powerful predictive value. Loss of stromal caveolin-1 in breast cancers is associated with early tumor recurrence, metastasis, and drug resistance, leading to poor clinical outcome